1. Field of the Invention
This invention relates to a multilayer analytical element. More particularly, this invention relates to an improvement of a multilayer analytical element containing a color indicator system for detection of hydrogen peroxide which is appropriately employable in quantitative analysis of hydrogen peroxide in a liquid sample, or in quantitative analysis of a specific component through quantitative determination of the amount of hydrogen peroxide produced upon contact of the specific component with an oxidase enzyme system.
2. Description of Prior Arts
A quantitative analysis of a specific component (referred to herein as "analyte") based on quantitative determination of the amount of H.sub.2 O.sub.2 produced by an oxidation reaction between the analyte or a reaction product of the analyte and oxidase, by means of an appropriate determination procedure has recently become more important. The reason is that the quantitative determination of H.sub.2 O.sub.2 can be done accurately and reliably, for instance, by colorimetric determination of a colored product formed by the action of H.sub.2 O.sub.2 in the presence of peroxidase.
As for the colorimetric determination method based on the above-mentioned principle, there is known a method using a reagent system proposed by P. Trinder (Ann. Clin. Biochem., 6, 24-27 (1969)). This method involves: producing H.sub.2 O.sub.2 by a reaction between an analyte and oxidase; causing an oxidative coupling reaction between 4-aminoantipyrine (or an analogue thereof) and a phenol (or a naphthol) in the presence of produced H.sub.2 O.sub.2 and peroxidase to produce a colored product; and quantitatively determining the so produced colored product. This reaction system is advantageous because the same detection system is employable regardless of varying the kind of oxidase. Accordingly, this reaction system has been studied for application in detection of various analytes. Examples of oxidases particularly important in the art of clinical chemical tests include glucose oxidase, cholesterol oxidase, uricase, glycerol oxidase, and phosphoglucose oxidase, etc.
An analytical element employing said oxidase and a detection system for the produced H.sub.2 O.sub.2 in the form of an integral multilayer analytical element, or a strip (such as a filter paper strip) impregnated with these reagents is widely employed for clinical tests. These analytical elements comprise a composition containing reagents directly participating in the detection of an analyte which is impregnated in a filter paper strip or the like, or coated over a filmy support.
The integral multilayer analytical element can be in a variety of constitutions, and different functional layers can be incorporated into the element under lamination as need arises. Examples of known functional layers include: a spreading layer for uniform spreading of a liquid sample (U.S. Pat. No. 3,992,158, GB No. 1 440 464, and U.S. Pat. No. 4,292,272); a light-shielding layer providing a light-scattering white surface for reflective measurement, as well as serving for optically separating the colored product produced upon the reaction from colored components contained within the upper layer (U.S. Pat. No. 3,992,158, GB No. 1,440,464, and U.S. Pat. Nos. 4,042,335, 4,255,384, and 4,292,274, etc.); a barrier layer for selective diffusion of component, product, etc. (U.S. Pat. No. Re. 30,267) or a liquid-blocking air barrier layer for the same purpose (Japanese Patent Provisional Publication No. 58(1983)-77660, and GB 2 114 737A); a detection layer for efficient detection by mordanting the colored product (U.S. Pat. Nos. 4,042,335 and 4,144,306); a migration inhibiting layer for preventing diffusion of the colored product into other layers (U.S. Pat. No. 4,166,093); and an adhesive layer for improvement of adhesion between the incorporated layers (U.S. Pat. No. 4,292,272, etc.).
As a result of the study of the present inventor, it has been noted that a calibration curve prepared for quantitative analysis of glucose or cholesterol using a multilayer analytical element containing in its reagent layer a color-forming reagent composition for detection of H.sub.2 O.sub.2 including glucose oxidase or a combination of cholesterol esterase and cholesterol oxidase, peroxidase, 4-aminoantipyrine, and 1,7-dihydroxynaphthalene shows a relatively gentle slope (.gamma.), and particularly gentle in a high concentration region where a liquid sample containing a great amount of glucose or cholesterol is calibrated. This means that such a analytical element gives poor accuracy in the quantitative analysis, as well as narrow measurable range.